This method allows cellular structure and function to be perturbed through the fusion of two different cell types to address questions such as how do cells respond to changes in organelle number. By imaging fluorescently tagged proteins within fused cells, cellular structures can be referenced back to their cell type of origin to study the mechanisms of organelle homeostasis and function. For fluorescent dye labeling, see the human cancer cell populations of interest such that each culture will have expanded to at least six times 10 to the sixth cells at a 70 to 90%confluency by the next morning.
On the day of labeling, wash the rootletin eGFP and rootletin-mScarlet cells two times with 10 milliliters of PBS per wash. Dilute Violet cell dye to 500 nanomolar in PBS and Far Red cell dye to 200 nanomolar in PBS. Label the rootletin eGFP cells with the diluted Violet cell dye and the rootletin-mScarlet cells with the diluted Far Red cell dye for one minute at room temperature.
At the end of the incubations, stop the reactions with 10 milliliters of DMEM for five minutes. Wash both labeled cell cultures and one unlabeled parental cell culture with 10 milliliters of fresh PBS before incubating all of the cultures with one milliliter of prewarmed trypsin. At the end of the incubation, transfer the detached cell solutions to individual 15 milliliter conical tubes for centrifugation and gently resuspend the dye labeled pellets in one milliliter of PBS and the unlabeled cells in one milliliter of DMEM.
Then return all of the cell samples to the cell culture incubator. For cell fusion experiment, mix 500 microliters of Violet dye labeled cells with 500 microliters of Far Red dye labeled cells in a new 15 milliliter tube and sediment the remaining unmixed labeled cells by centrifugation. Resuspend the pellets in one milliliter of fresh DMEM and place the cells back into the incubator.
Collect the mixed cells by centrifugation and add 700 microliters of 50%1450 PEG in a drop-wise manner to the pellet over a period of 30 seconds. After 3.5 minutes at room temperature, add 10 milliliters of serum-free DMEM drop-wise over a period of 30 seconds and place the tubes into the incubator for 10 minutes. At the end of the incubation, sediment the mixed cells by centrifugation and gently resuspend the pellet in one milliliter of complete medium.
To enrich for the fused cell population by FACS, gently filter all of the cells into individual FACS tubes through a 70 micrometer strainer and create forward scatter versus side scatter and doublet discrimination plots in the cytometer software. Run the unlabeled cells to record their fluorescence intensity and create gates to identify the fused cells. Next, run the single positive Violet dye labeled cells to confirm that there is no spillover of Violet signal into the Far Red channel and the single positive Far Red dye labeled cells to confirm that there is no Far Red signal in the Violet channel.
Then briefly run the fusion sample to confirm that the fused cells are visible with these gates. When the sorting parameters have been set, align the sorting stream centrally into an eight-well imaging dish containing 100 microliters of growth medium and sort the double positive fused cells directly into the dish. When all of the cells have been sorted, immediately place the dish into the cell culture incubator for two to 16 hours.
For immunofluorescent staining, first replace the cell culture supernatant with 200 microliters of 4%paraformaldehyde for a 15-minute incubation at room temperature. At the end of the fixation, wash the cells three times with 200 microliters of PBS per wash before permeabilizing the cells in 200 microliters of 0.1%nonionic surfactant for 10 minutes at room temperature. Next, block the nonspecific binding with 200 microliters of 3%bovine serum albumin in PBS for 30 minutes at room temperature followed by labeling with the antibodies of interest in 150 microliters of staining buffer.
After one hour at room temperature, wash the cells two times in 300 microliters of PBS for five minutes per wash. After the last wash, replace the wash with 200 microliters of fresh PBS. To acquire images of the fused cells, use an appropriate fluorescence microscope capable of four-color imaging and collect three-dimensional images.
Appropriately labeled cells are visible during flow cytometry by fluorescent signal at a higher level than unlabeled control cells. The gates can be set for sorting to enrich for the double positive cell population directly into imaging dishes for further microscopic analysis. Fusion induces a major rearrangement of the cellular architecture through the mixing of two cells into one.
Heterokaryons are identified as cells containing both fluorescent dye signals mixed inside a single cell without intervening plasma membranes. Additionally, two nuclei may also be visible in fused cells by Brightfield imaging. The cell structure and function may be further investigated through the merging of cells containing meGFP and mScarlet tagged proteins.
Fusion results in a doubling of the centrosome number inside heterokaryons visible as at least four pericentriolar material foci when the centrosome pericentriolar component NEDD1 is fluorescently tagged. The fusion of cells with endogenously fluorescently tagged rootletin allows the cell of origin of each centrosome to be identified in a heterokaryon. This protocol demonstrates how to fuse differentially labeled cell lines and how to image their organelles to understand their structure and function.
This robust protocol is both relatively easy and relatively inexpensive to perform compared to similar methods. This technique can be used to ask a range of questions such as how is organelle fusion regulated and how the cells respond to changes in subcellular organelle number.
